Vibrio parahaemolyticus is the most common bacterial cause of seafood-associated gastroenteritis worldwide. Despite the potential severity of V. parahaemolyticus infection, the majority of cases go unreported; however, a confluence of environmental and chronic medical factors will likely increase the prevalence, severity, and overall public health impact of V. parahaemolyticus infection in the future. The recently developed infant rabbit model of V. parahaemolyticus infection has transformed the study of this clinically important yet relatively understudied pathogen. Experiments in the infant rabbit model demonstrated the essential role of T3SS2, a type III secretion system that injects bacterial proteins into host cells, in colonization of the mammalian gastrointestinal tract. Besides T3SS2, there is virtually no knowledge of additional genes that enable this global pathogen to colonize the host intestine, proliferate, and cause disease. I conducted a genome-wide transposon-insertion sequencing (TIS) screen to identify V. parahaemolyticus genetic loci necessary for infection and identified 164 genes likely to play a role in the pathogenicity of this organism. The goal of this proposal is to validate a compelling subset of these `conditionally essential' (CE) genes, which are necessary for growth in the intestine but dispensable for growth in the laboratory. The studies outlined in Aim 1 will test the hypothesis that 6 CE genes, encoding hypothetical proteins within the T3SS2 gene cluster, act as undefined components of T3SS2, the primary V. parahaemolyticus virulence factor. The studies outlined in Aim 2 will test the hypothesis that a recently discovered `translocation and assembly module' (Tam) is necessary for the secretion or outer membrane localization of undefined V. parahaemolyticus virulence factors. These experiments will greatly expand our understanding of the factors and mechanisms that enable V. parahaemolyticus pathogenicity. Ultimately, elucidating the mechanisms by which these conditionally essential genes contribute to V. parahaemolyticus intestinal colonization and virulence will help guide efforts to develop new therapeutics for enteric diseases and to better understand host-pathogen interactions within the gastrointestinal tract.